1. Field of the Invention
The invention concerns diagnostic procedures performed on biological fluids and more particularly concerns a means and method of determining total and direct bilirubin concentrations in blood serum or plasma, employing diazonium salt reagents.
2. Brief Description of the Prior Art
Colorimetric techniques for the determination of bilirubin in blood were known prior to this invention; see for example Winsten et al., Clinical Chem., 14, (8), pps 850-1, (1968); Walters et al., Microchem. J., 13:253, (1968); Walters et al., Microchem. J., 15:231, (1970); U.S. Pat. Nos. 3,754,862 and 2,737,501. Although the prior art techniques include the use of diazonium salt reagents solubilized with dimethyl sulfoxide (see U.S. Pat. No. 3,511,607), such techniques have required performance by technical personnel having a fairly high degree of technical proficiency. One of the difficulties in total and direct bilirubin determinations employing as the reagent diazonium salts resides in the fact that such reagents are relatively unstable; see for example U.S. Pat. No. 2,737,501. Therefore, the reagent should be freshly prepared immediately before use in carrying out a blood analysis if accuracy is desired; see U.S. Pat. No. 3,652,222 which recommends that such reagents be used within 1 hour of preparation. Not only is a degree of technical proficiency required for the accurate preparation of a reliable reagent, but immediate-before-use preparation requires additional time and work on the part of the technician prior to his or her actually carrying out an analysis of an unknown specimen of blood serum or plasma.
The analysis kit of the present invention provides a means whereby a technician is provided with diazonium salt reagent precursors in such form that the reagent may be immediately prepared with a minimum of effort. The precursor reactants and consequently the kit per se is stable for a period of at least two years when stored out of direct sunlight and at room temperatures (below 86.degree. F.). Furthermore, the analysis kit according to the method of this invention provides for the uniform preparation of control reagents. The kit which forms part of this invention also reduces the complexity of total and direct bilirubin analysis procedure so that it may be carried out by technicians having relatively little experience and training. The results of an analysis carried out employing the kit of the invention are uniform and compare remarkably well with more complex methods of determining bilirubin.
Bilirubin occurs in the blood in two forms, first in the free form or unconjugated and secondly as bilirubin glucuronide or conjugated bilirubin. Unconjugated bilirubin is formed as a decomposition product of erythrocytes and is conjugated and excreted into the bile by the liver. Conjugated bilirubin is measured as direct reading bilirubin and unconjugated bilirubin is quantitated by measuring total bilirubin and then subtracting direct reading bilirubin.
The method of the invention is particularly advantageous in that it permits for a highly accurate determination of total and direct bilirubin, with the expenditure of a minimum amount of time and effort on the part of technical personnel.